Fumihiro Ishikawa

Gene expression profiling identifies a role for CHOP during inhibition of the mitochondrial respiratory chain.

Mitochondrial dysfunction, in particular, interference in the respiratory chain, is often responsible for the toxicogenic effects of xenobiotics. In this study, changes in gene expression resulting from pharmacological inhibition of the respiratory chain were studied by DNA microarray analysis using cells treated with rotenone or antimycin A, which inhibit complexes I and III of the electron transport system, respectively. Forty-eight genes were either up- or down-regulated more than 3-fold. These included stress- and/or metabolic-related effector genes and several transcriptional regulators represented by CHOP-10. Further study using siRNA showed that among the four genes studied, up-regulation of three was dependent on CHOP-10. C/EBPbeta, a dimerizing partner of CHOP-10, was also involved in two of the three genes including Trib3, implying that CHOP-10, heterodimerizing with C/EBPbeta or another partner played a key role in the expression of a set of genes under stress. Although CHOP-10 and Trib3 were both ER-stress response genes, signal inducing Trib3 during mitochondrial stress was distinct from that during ER stress. Cytotoxicity caused by inhibition of the respiratory chain was attenuated by treatment with siRNA for CHOP-10. This study demonstrated the importance of CHOP-10 in coordinating individual gene expression in response to the mitochondrial stress.

Importance of mitochondrial dysfunction in oxidative stress response: A comparative study of gene expression profiles

Abstract Mitochondria are considered to play an important role in oxidative stress response since they are a source of reactive oxygen species and are also targeted by these species. This study examined the mitochondrial conditions in cells of epithelial origin that were exposed to H2O2 and found a decline in the membrane potential along with a specific loss of UQCRC1, a sub-unit of complex III, suggesting that mitochondrial dysfunction occurs upon exposure to oxidative stress. This observation led to the hypothesis that certain cellular responses to oxidative stress occurred because of mitochondrial dysfunction. When mitochondria-less (pseudo ρ0) cells were examined as a model of mitochondrial dysfunction, striking similarities were found in their cellular responses compared with those found in cells exposed to oxidative stress, including changes in gene expression and gelatinolytic enzyme activities, thus suggesting that cellular responses to oxidative stress were partly mediated by mitochondrial dysfunction. This possibility was further validated by microarray analysis, which suggested that almost one-fourth of the cellular responses to oxidative stress were mediated by mitochondrial dysfunction that accompanies oxidative stress, thereby warranting a therapeutic strategy that targets mitochondria for the treatment of oxidative stress-associated diseases.

A mitochondrial thioredoxin-sensitive mechanism regulates TGF-β-mediated gene expression associated with epithelial-mesenchymal transition.

Transforming growth factor (TGF)-β is a pro-oncogenic cytokine that induces the epithelial-mesenchymal transition (EMT), a crucial event in tumor progression. During TGF-β-mediated EMT in NMuMG mouse mammary epithelial cells, we observed sustained increases in reactive oxygen species (ROS) levels in the cytoplasm and mitochondria with a concomitant decrease in mitochondrial membrane potential and intracellular glutathione levels. In pseudo ρ0 cells, whose respiratory chain function was impaired, the increase in intracellular ROS levels was abrogated, suggesting an important role of mitochondrial activity as a trigger for TGF-β-stimulated ROS generation. In line with this, TGF-β-mediated expression of the EMT marker fibronectin was inhibited not only by chemicals that interfere with ROS signaling but also by exogenously expressed mitochondrial thioredoxin (TXN2) independent of Smad signaling. Of note, TGF-β-mediated induction of HMGA2, a central mediator of EMT and metastatic progression, was similarly impaired by TXN2 expression, revealing a novel mechanism involving a thiol oxidation reaction in mitochondria, which regulates TGF-β-mediated gene expression associated with EMT.

Competitive Nuclear Export of Cyclin D1 and Hic-5 Regulates Anchorage Dependence of Cell Growth and Survival

Anchorage dependence of cell growth and survival is a critical trait that distinguishes nontransformed cells from transformed cells. We demonstrate that anchorage dependence is determined by anchorage-dependent nuclear retention of cyclin D1, which is regulated by the focal adhesion protein, Hic-5, whose CRM1-dependent nuclear export counteracts that of cyclin D1. An adaptor protein, PINCH, interacts with cyclin D1 and Hic-5 and potentially serves as an interface for the competition between cyclin D1 and Hic-5 for CRM1. In nonadherent cells, the nuclear export of Hic-5, which is redox-sensitive, was interrupted due to elevated production of reactive oxygen species, and cyclin D1 was exported from the nucleus. When an Hic-5 mutant that was continuously exported in a reactive oxygen species-insensitive manner was introduced into the cells, cyclin D1 was retained in the nucleus under nonadherent conditions, and a significant population of cells escaped from growth arrest or apoptosis. Interestingly, activated ras achieved predominant cyclin D1 nuclear localization and thus, growth in nonadherent cells. We report a failsafe system for anchorage dependence of cell growth and survival.

Downregulation of hepatocyte nuclear factor-4α and its role in regulation of gene expression by TGF-β in mammary epithelial cells

We found that a specific isoform of hepatocyte nuclear factor 4alpha (HNF-4alpha), HNF-4alpha8, was expressed in mouse mammary epithelial NMuMG cells, and that its expression was repressed by TGF-beta. The repression was interfered by dominant negative forms of activin receptor-like kinase 5 (ALK5) and Smad3, and sensitive to cycloheximide, suggesting the involvement of additional protein(s) as well as ALK5 and Smad3 in the repression. Further study showed that high mobility group A2 (HMGA2), which is reported to be directly upregulated by Smads, repressed HNF-4alpha8 expression. Therefore, it is likely that HMGA2 mediates the downregulation of HNF-4alpha8 downstream of ALK5 and Smads To determine the significance of the downregulation of HNF-4alpha8 in TGF-beta signaling, we performed DNA microarray analysis and extracted a subgroup of TGF-beta1-regulated genes, including tenascin C and tissue inhibitor of metalloproteinase 3 (TIMP-3), whose regulation by TGF-beta1 was attenuated by forced expression of HNF-4alpha8. HMGA2 has recently emerged as a transcriptional organizer of TGF-beta signaling, regulating several key factors involved in epithelial-mesenchymal transition (EMT). In this study, we identified an isoform of HNF-4alpha as a new target downstream of HMGA2 and assigned a new role to HNF-4alpha in the TGF-beta signaling/transcriptional cascade driven by ALK5/Smad/HMGA2 and associated with the malignant transformation of cells.

Prognostic impact of the number of viable circulating cells with high telomerase activity in gastric cancer patients: A prospective study

The identification of circulating tumor cells (CTCs) in peripheral blood is a useful approach to estimate prognosis, monitor disease progression and measure treatment effects in several types of malignancies. We have previously used OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene. GFP-positive cells (GFP+ cells) were counted under a fluorescence microscope. Our results showed that the number of at least 7.735 µm in diameter GFP+ cells (L-GFP+ cells) in the peripheral blood was a significant marker of prognosis in gastric cancer patients. However, tumor cells undergoing epithelial-mesenchymal transition (EMT) have been reported to be smaller in size than cells without EMT features; thus, CTCs undergoing EMT may escape detection with this technique. Therefore, in this study, we analyzed the relationship between patient outcome and the number of GFP+ cells of any size. We obtained peripheral blood samples from 65 patients with gastric cancer. After infection of OBP-401, GFP+ cells were counted and measured. The relationship between the number of GFP+ cells and surgical outcome was analyzed. The median follow-up period of the surviving patients was 36 months. A significant difference in overall survival was found between patients with 0-5 and patients with ≥6 L-GFP+ cells. No clear relationship was established between the number of small-sized GFP+ cells and patient prognosis. The number of L-GFP+ cells was significantly related to overall survival in patients with gastric cancer. The detection of L-GFP+ cells using OBP-401 may be a useful prognostic marker in gastric cancer.

A HIC-5- and KLF4-dependent Mechanism Transactivates p21Cip1 in Response to Anchorage Loss

Background: Anchorage dependence of cell growth remains incompletely understood. Results: A cyclin-dependent kinase inhibitor (p21Cip1) is transcriptionally up-regulated on anchorage loss, depending on Kruppel-like factor 4 (KLF4) and a molecular scaffold of hydrogen peroxide-inducible clone-5 (HIC-5). Conclusion: On anchorage loss, HIC-5 localizes at the nuclear matrix and promotes KLF4 tethering to DNA. Significance: A novel mechanism regulating gene expression in a detachment-dependent manner has emerged. Anchorage loss elicits a set of responses in cells, such as transcriptional changes, in order to prevent inappropriate cell growth in ectopic environments. However, the mechanisms underlying these responses are poorly understood. In this study, we investigated the transcriptional up-regulation of cyclin-dependent kinase inhibitor p21Cip1 during anchorage loss, which is important for cell cycle arrest of nonadherent cells in the G1 phase. Up-regulation was mediated by an upstream element, designated as the detachment-responsive element (DRE), that contained Kruppel-like factor 4 (KLF4) and runt-related transcription factor 1 (RUNX1) recognition sites; both of these together were necessary for transactivation, as individually they were insufficient. RNAi experiments revealed that KLF4 and a multidomain adaptor protein, hydrogen peroxide-inducible clone 5 (HIC-5), were critically involved in DRE transactivation. The role of HIC-5 in this mechanism was to tether KLF4 to DNA sites in response to cellular detachment. In addition, further analysis suggested that oligomerization and subsequent nuclear matrix localization of HIC-5, which was accelerated spontaneously in cells during anchorage loss, was assumed to potentiate the scaffolding function of HIC-5 in the nucleus and consequently regulate p21Cip1 transcription in a manner responding to anchorage loss. At the RUNX1 site, a LIM-only protein, CRP2, imposed negative regulation on transcription, which appeared to be removed by anchorage loss and contributed to increased transcriptional activity of DRE together with regulation at the KLF4 sites. In conclusion, this study revealed a novel transcriptional mechanism that regulated gene expression in a detachment-dependent manner, thereby contributing to anchorage-dependent cell growth.

Critical roles of the cAMP-responsive element-binding protein-mediated pathway in disorganized epithelial phenotypes caused by mitochondrial dysfunction

In most human cancers, somatic mutations have been identified in the mtDNA; however, their significance remains unclear. We recently discovered that NMuMG mouse mammary epithelial cells, when deprived of mitochondria or following inhibition of respiratory activity, undergo epithelial morphological disruption accompanied with irregular edging of E‐cadherin, the appearance of actin stress fibers, and an altered gene expression profile. In this study, using the mtDNA‐less pseudo ρ0 cells obtained from NMuMG mouse mammary epithelial cells, we examined the roles of two mitochondrial stress‐associated transcription factors, cAMP‐responsive element‐binding protein (CREB) and C/EBP homologous protein‐10 (CHOP), in the disorganization of epithelial phenotypes. We found that the expression of matrix metalloproteinase‐13 and that of GADD45A, SNAIL and integrin α1 in the ρ0 cells were regulated by CHOP and CREB, respectively. Of note, knockdown and pharmacological inhibition of CREB ameliorated the disrupted epithelial morphology. It is interesting to note that the expression of high mobility group AT‐hook 2 (HMGA2), a non‐histone chromatin protein implicated in malignant neoplasms, was increased at the protein level through the CREB pathway. Here, we reveal how the activation of the CREB/HMGA2 pathway is implicated in the repression of integrin α1 expression in HepG2 human cancer cells, highlighting the importance of the CREB/HMGA2 pathway in malignant transformation associated with mitochondrial dysfunction, thereby raising the possibility that the pathway indirectly interferes with the cell–cell adhesion structure by influencing the cell–extracellular matrix adhesion status. Overall, the data suggest that mitochondrial dysfunction potentially contributes to neoplastic transformation of epithelial cells through the activation of these transcriptional pathways.

Linkage of E2F1 transcriptional network and cell proliferation with respiratory chain activity in breast cancer cells

Mitochondria are multifunctional organelles; they have been implicated in various aspects of tumorigenesis. In this study, we investigated a novel role of the basal electron transport chain (ETC) activity in cell proliferation by inhibiting mitochondrial replication and transcription (mtR/T) using pharmacological and genetic interventions, which depleted mitochondrial DNA/RNA, thereby inducing ETC deficiency. Interestingly, mtR/T inhibition did not decrease ATP levels despite deficiency in ETC activity in different cell types, including MDA‐MB‐231 breast cancer cells, but it severely impeded cell cycle progression, specifically progression during G2 and/or M phases in the cancer cells. Under these conditions, the expression of a group of cell cycle regulators was downregulated without affecting the growth signaling pathway. Further analysis suggested that the transcriptional network organized by E2F1 was significantly affected because of the downregulation of E2F1 in response to ETC deficiency, which eventually resulted in the suppression of cell proliferation. Thus, in this study, the E2F1‐mediated ETC‐dependent mechanism has emerged as the regulatory mechanism of cell cycle progression. In addition to E2F1, FOXM1 and BMYB were also downregulated, which contributed specifically to the defects in G2 and/or M phase progression. Thus, ETC‐deficient cancer cells lost their growing ability, including their tumorigenic potential in vivo. ETC deficiency abolished the production of reactive oxygen species (ROS) from the mitochondria and a mitochondria‐targeted antioxidant mimicked the deficiency, thereby suggesting that ETC activity signaled through ROS production. In conclusion, this novel coupling between ETC activity and cell cycle progression may be an important mechanism for coordinating cell proliferation and metabolism.

Change in number and size of circulating tumor cells with high telomerase activity during treatment of patients with gastric cancer

Detection of circulating tumor cells (CTCs) in peripheral blood is useful for estimating the prognosis of patients with cancer. We previously reported the detection of CTCs by OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene. We demonstrated that the number of large (L)-GFP+ cells (≥7.735 µm in diameter) in peripheral blood samples correlated significantly with the prognosis of treatment-naïve gastric cancer patients, whereas the number of small (S)-GFP+ cells (<7.735 µm in diameter) did not. In the present study, we studied the change in the number of GFP+ cells during treatment, and analyzed the association between the number of GFP+ cells in blood samples and the outcome of patients. Peripheral blood samples were obtained from 37 gastric patients prior and subsequent to surgery (three samples per time point). Upon infection of blood cells with OBP-401, GFP+ cells of different sizes were counted and measured. The association between the number of GFP+ cells and surgical outcome was determined by statistical analysis. The median follow-up period after surgery was 39 months. Although the difference was not significant, patients with ≥6 L-GFP+ cells in preoperative blood samples had a lower relapse-free survival rate than patients with 0-5 L-GFP+ cells. There was no significant correlation between the number of L-GFP+ cells in postoperative blood samples and the prognosis of patients receiving adjuvant therapy. Although the difference was not significant, the number of S-GFP+ cells in samples from patients who had received postoperative chemotherapy was higher than in those who had not. The number of L-GFP+ cells was not significantly correlated with the relapse-free survival rate in gastric cancer patients who underwent surgery. The number of S-GFP+ cells was relatively high in samples from patients who had received postoperative chemotherapy.